Method of causing selective immunosuppression using HL-60-related lectins

ABSTRACT

Pharmaceutical compositions useful in the treatment of autoimmune conditions include as an active ingredient a soluble lectin having a molecular weight of about 14 kilodaltons or a fragment thereof. The lectin or fragment binds β-galactoside-containing moieties independent of the presence or absence of Ca +2 , stimulates hemagglutination of trypsinized rabbit erythrocytes in standard lectin assays wherein the stimulation is inhibited by lactose or thiogalactoside, has an amino acid sequence containing at least one N-glycosylation site and is at least 90% homologous to the amino acid sequence shown in positions 2-135 of FIG.  1  or the relevant portions thereof. The composition is used for treatment of autoimmune conditions such as rheumatoid arthritis, myasthenia gravis, and multiple sclerosis, as well as modulating the immune response in an allergic reactions or to organ or tissue transplant rejection. The inventive composition can be combined with general immunosuppressants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation applicaton of Ser. No. 08/719,866,now U.S. Pat. No. 6,153,195 filed Sep. 25, 1996, which is a divisionalof Ser. No. 08/326,739 now U.S. Pat. No. 5,693,760 filed Oct. 20, 1994,which is a continuation of Ser No. 07/976,928 now abandoned filed Nov.16, 1992 continuation-in-part of U.S. patent application Ser. No.07/313,649, now abandoned filed Feb. 21, 1989, which is acontinuation-in-part of U.S. patent application Ser. No. 263,734, nowabandoned filed Oct. 28, 1988, which is, in turn, a continuation-in-partof U.S. patent application Ser. No. 181,747, now abandoned filed Apr.14, 1988 each of which are incorporated herein by reference in theirentirety and to which we claim priority under 35 U.S.C. §120.

TECHNICAL FIELD

The invention relates to the use of carbohydrate-binding proteins asregulators of cell differentiation and immunity. In particular, itconcerns a pharmaceutical composition where the active ingredient is asoluble lectin of about 14 kD or a fragment thereof which can beisolated from human HL-60 cells or placenta tissue. Recombinantmaterials and methods to produce these inventive lectins arealsoprovided. This invention is also directed to methods to treatautoimmune diseases and to prevent transplant rejection.

BACKGROUND ART

Lectins are defined as proteins which specifically bind carbohydrates qfvarious types. Initial interest was focused on those isolated fromplants such as concanavalin A and ricin agglutinin. These lectins, itwas found, were useful in protein purification procedures due to theglycosylation state of a number of proteins of interest. Among thesoluble lectins, there appear to be a number of varieties with varyingmolecular weights and/or carbohydrate specificities. Sparrow, C. P., etal., J. Biol. Chem. (1987) 252:7383-7390 describe three classes ofsoluble lectins from human lung, one of 14 kD, one of 22 kD, and a thirdof 29 kD. All of theselectins are specific to β-D-galactosides. Thecarbohydrate specificities of the 14 kD class are for the most partsimilar, but the larger molecular weight species tend to have differentspecificities. Other species are also noted as showing more than onesoluble β-D-galactoside-binding lectin, including mouse (Roff, C. F., etal., J. Biol. Chem. (1983) 258:10637-10663); rat (Cerra, R. F., et al.,J. Biol. Chem. (1985) 260:10474-10477) and chickens (Beyer, E. C., etal., J. Biol. Chem. (1980) 255:4236-4239). Among the variousβ-D-galactoside-specific soluble lectins, ligand specificity isconsiderably different, and the approximately 14 kD group appearsdistinct from the 22 kD and 29 kD representatives described by Sparrow,et al., supra.

Recently, however, interest has focused on a group oflactose-extractable lectins which bind specifically to certainβ-D-galactoside containing moieties and are found in a wide range ofmammalian, in-vertebrate, avian, and even microbial sources. All of thelectins in this class appear to contain subunits with molecular weightsof about 12-18 kD. Furthermore, these lectins can be readily classifiedby virtue of a simple diagnostic test: their ability to agglutinatetrypsin-treated rabbit red blood cells is specifically inhibited bycertain β-D-galactose-containing moieties. Thus, although the lectinsthemselves agglutinate trypsinized rabbit erythrocytes, theagglutination can be inhibited by, for example, lactose,thiodigalactoside and certain other β-D-galactose containing moieties.Other common characteristics include no requirement for metal ions ineffecting agglutination and the required presence of a reducing agentsuch as a thiol.

Gitt, M. A. et al., Proc. Natl. Acad. Sci. USA (1986) 83:7603-7607obtained two cDNA clones from immunoscreening a human hepatoma cDNAlibrary with an antiserum specific to a human lung lectin. Gitt et al.partially sequenced the cDNAs and the lectins. Gitt compared thesesequences with that of the human lung chicken lectin. Although therewere marked similarities with chicken and lung lectin, Gitt et al.concluded “In contrast with lung [encoding one form of HL-14 lectin],human hepatoma appears to express two other forms of HL-14” (page 7607).Kasai, K. et al., in Japanese Kokai 60/184020 describe a human placentallectin of approximately 14 kD. The sequence of this placental lectin wasshown by the same group to be somewhat similar to that isolated fromchick tissues (Ohyama, Y., et al., Biochem. Biophys. Res. Commun. (1986)134:51-56). The chick-derived lectin was shown to be similar instructure to that of discoidin I, which is a lectin also observed duringcertain developmental stages of the cellular slime mold Dictyosteliumdiscoideum.

Caron, M., et al., Biochim. Biophys. Acta (1987) 925:290-296 describethe purification and characterization of lectins from rat and bovinebrain tissue. deCabutti, N. E. F., et al., FEBS Letters (1987)223:330-334 describe a lectin from amphibian ovary. The isolation fromeel of a similar “electrolectin” had previously been described by Levi,G., et al., J. Biol. Chem. (1981) 256:5735-5740. An additional analogous14 kD lectin was produced by cloning and expression of cDNA derived fromvarious murine fibrosarcoma cell lines by Raz, A., et al., ExperimentalCell Research (1987) 173:109-116. A rat lung 14 kD lectin, and the cDNAencoding it were described by Clerch, L. B., et al., Biochemistry (1988)27:692-699. Joubert, R., et al., Develop. Brain Res. (1987) 36:146-150describe the isolation of lectins from rat brain which are capable ofagglutinating brain cells. Raz, A., et al., Cancer Res. (1981)41:3642-3647 describe a variety of lectins from neoplastic cells ofvarious mammalian species.

Paroutaud, P., et al.,(Proc. Natl. Acad. Sci. USA (1987) 84:6345-6348)compared the amino acid sequences of several animal lectins includingthose from chick, eel, human placenta, human lung, and twohepatoma-derived lectins (all of these lectins described as referencedabove). Only the chicken lectin contains an “N-linked” glycosylationsite, which is not conjugated to saccharide. No known mammalian lectinin this family has an N-linked glycosylation site.

Although several of the above references disclose some structuralsimilarities with the present invention, none of the references teachthe same bioactivity of the unique lectin of the present invention.

The preferred lectins of the present invention are isolated from thehuman promyelocytic leukemia cell line HL-60 or human placenta tissue.Lectins have been isolated from the HL-60 cell line by others, but theyare markedly different from the lectins of the present invention.Paietta, E., et al., Cancer Res. (1988) 48:280-287 describe amembrane-bound (not soluble), 17 kd lectin which recognizes N-acetylneuraminic acid as well as galactose terminating biantennaryoligosaccharide structures. Unlike other 14 kd lectins, this 17 kdlectin is not inhibited by complex galactose saccharides such asthiodigalactoside and does not require reducing thiol groups for bindingactivity.

Thus, ligand specificity and biodistribution of the lectin proteindescribed herein are an abrupt departure from the earlier disclosedlectins.

Because the activities of lectins in regulating the iune system andmediating other forms of intercellular commumication are so subtle innature and so critically tuned to the host environment, subtle changesin structure can result in a wide range of such regulators withdiffering therapeutic and diagnostic uses. As described above, a numberof members of the class of β-D-galactose-binding soluble lectinsweighing approximately 14 kD are known in the art. However, while theselectins have some similarities, they are not interchangeabletherapeutically or diagnostically. In addition, it appears that forlectins which can be glycosylated, the extent and nature of theglycosylation can be manipulated to alter important lectin properties(e.g., circulating half-life, metabolism in vivo, solubility, stability,and specific activity).

Levy et al. (Eur. J. Immunol. (1983) 13:500-507) reported thatelectrolectin binds to peripheral blood and lymph node lymphocytes andis mitogenic. When Levy et al. administered electrolectin to rabbitssimultaneously with acetylcholine receptor, it prevented the developmentof a myasthenia gravis-like condition. Administering electrolectin afterdevelopment of myasthenia gravis caused complete recovery, in spite ofhigh antibody levels specificfor the acetylcholine receptor. Becauseelectrolectin did not interfere with acetylcholine interaction with itsreceptor, Levy et al. proposed that electrolectin had an effect on theimmune system.

Prominent diseases in which there is an immune system dysfunctioninclude autoimmune diseases such as myasthenia gravis (MG), rheumatoidarthritis (RA) systemic lupus erythematosus (SLE), multiple sclerosis(MS) and juvenile arthritis. Typically MG, RA, SLE and MS are treatedfirst with corticosteroids. Steroidal drugs have been used for decadesand their adverse effects arewell known. Adverse effects that can beanticipated in all patients on prolonged steroid therapy includeosteoporosis, truncal obesity, impaired wound healing, infections andgrowth arrest in children. Less frequently occurring adverse effectsinclude myopathy, hypertension, hyperlipidemia, diabetes mellitus andcataracts. Severe side effects may develop and require patientmonitoring. These include glaucoma, intracranial hypertension,intestinal perforation, and ulcers.

If MG, RA, SLE or MS become refractory to steroids, then increasinglytoxic drugs are employed, including azathioprine, methotrexate andcyclophosphamide. The primary effect of azathioprine is inhibiting DNAsynthesis, thus lowering numbers of T and B lymphocytes. In addition,azathioprine inhibits the mixed lymphocyte reaction and immunoglobulinproduction, but does not consistently affect delayed-typehypersensitivity. The major adverse effect of azathioprine ispancytopenia, particularly lymphopenia and granulocytopenia.Consequently, there are increased risks of viral, fungal, mycobacterialand protozoal infections. An increased rate of lymphoreticularmalignancies has been reported in kidney transplant patients, but not inpatients with RA.

Methotrexate inhibits folic acid synthesis and is cytotoxic, suppressingbone marrow. At the low doses used for RA, methotrexate should notdecrease the numbers of lymphocytes; but IgM and IgG are reduced. Sideeffects include pneumonia, nausea, stomach upsets, mouth ulcers,leukopenia, throubocytopenia, and a form of hepatic fibrosis, which canonly be diagnosed by liver biopsy.

Cyclophosphamide is also used in RA therapy. It is metabolized in theliver to a compound which cross-links DNA. Cyclophosphamide iscytotoxic, with severe toxicity seen even at low doses. It affects RA byreducing numbers of B- and T-lymphocytes, decreasing the immunoglobulinconcentrations and diminishing B-cell responsiveness to mitogenicstimuli. Hair loss, infections, and powerful nausea are common. Withprolonged administration, patients develop malignancies at an increasedrate.

Cyclosporin does not suppress white cells, but it is a powerfulimmunomodulatory drug and is effective in treating rheumatoid arthritis.However, an important side effect is renal toxicity.

Monoclonal antibodies to CD4 have been used in autoimmune diseases, butthey cause nonspecific immunosuppression. It has been recommended thatnew therapies interfere with the initial presentation of specificinciting antigens to T-lymphocytes. (Wraith et al., Cell (1989)57:709-715).

Other drugs have been used specifically in RA, including gold salts,antimalarials, sulfasalazine and penicillamine. Gold salts are givenintramuscularly and their effect may not be seen for months. Adverseeffects of gold treatment include bone marrow aplasia,glomerulonephritis, pulmonary toxicity, vasomotor reactions andinflammatory flare. Antimalarials exert several effects on the immunesystem without decreasing the numbers of lymphocytes. The most seriousside effects of antimalarials include retinal pigment deposition, rashand gastrointestinal upset. Sulfasalazine has several effects whichcontribute to its effect on RA; however, it has numerous side effects.Penicillamine has been successfully used in RA; however, its numerousside effects have limited its use. Penicillamine has been reported tocause other autoimmune diseases, including myasthenia gravis and SLE.

When patients receive allografts (transplanted tissue from other humansor other sources), their immune systems can destroy the allografts inshort order were it not for the administration of immunosuppressantdrugs. A number of different organs and tissues are now transplanted,including the kidneys, heart, lungs, skin, bone marrow, cornea, andliver. Drugs frequently used in transplant patients include cyclosporin,azathioprine, rapamycin, other macrolides such as FK506, prednisone,methylprednisolone, CD4 antibodies and cyclophosphamide. Frequentlythese drugs must be given in higher doses and for longer periods totransplant patients than to patients with autoimmune diseases. Hence,side effects from these drugs (discussed above) may be more common andsevere in transplant patients.

What was needed before the present invention is a drug that wouldselectively treat autoimmune diseases and transplant rejection withoutthe severe side effects of the previously known therapies.

SUMMARY OF THE INVENTION

The present invention is directed to a pharmaceutical composition. Theactive agent is a soluble lectin of MW of about 14 kD or a fragmentthereof, wherein the lectin or fragment (1) bindsβ-galactoside-containing moieties whether Ca⁺² is present or not, (2)stimulates hemagglutination of trypsinized rabbit erythrocytes instandard lectin assays which is inhibited by lactose orthiodigalactoside, (3) provides an amino acid sequence containing atleast one N-glycosylation site and at least 90% homologous to the aminoacid sequence shown in positions 2-135 of FIG. 1 or the relevantportions thereof, and wherein the active ingredient is mixed with acarbohydrate and at least one pharmaceutically acceptable excipient. Thecomposition can also contain one or more general immune systemsuppressants such as cyclosporin. The invention also providesrecombinant materials and methods to produce these new lectins.

In other aspects, the invention is directed to methods to treatautoimmune diseases and to prevent transplant rejection. The efficacy ofthese methods results from the surprising ability of the inventivelectin to suppress the host imme response to both autoimmunogens andforeign tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cDNA sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of both the HL-60 and placenta lectin.Superscript numbers correlate to the corresponding nucleotides and boxesshow start and stop codons. The asterisk indicates the possible N-linkedglycosylation site.

FIG. 2 shows immunostaining of thymic epithelium by anti-lectin andanti-cytokeratin antibodies.

FIG. 3 shows an electrophoresis gel with staining in a locationindicative of mRNA for the inventive lectin in thymic epithelial cells.

FIG. 4 shows the percentage of ARR T-cells that adhered to thymicepithelial cells with and without enzyme treatment.

FIG. 5 shows results of hemagglutinin bioassay of purified lectins fromhuman placenta, HL-60 cells and E. coli cells transfected with vectorscontaining lectin cDNA and an operably linked secretion signal, andinhibition of agglutination by sugars.

FIG. 6 shows the effect of several doses of the inventive lectin on theprimary immune response to tetanus toxoid.

FIG. 7 shows the effect of several doses of the inventive lectin on theprimary immune response to pneumococcal polysaccharide.

FIG. 8 shows the effect of several doses of the inventive lectin on theprimary immune response against the Torpedo receptor.

FIG. 9 shows the effect of several doses of the inventive lectin on theprimary immune response against mouse skeletal muscle receptor inanimals immunized with the Torpedo receptor.

FIGS. 10 and 11 show the results of the treatment with the inventivelectin on clinical symptoms in a rat model for multiple sclerosis.

FIG. 12 is a graphical representation of the effect of recombinantlyproduced lectin in a rat model for multiple sclerosis.

FIG. 13 shows the effect of the recombinantly produced lectin on arelapse murine model for multiple sclerosis.

FIG. 14 is a graphical comparison of the survival of skin allograftswhenrats received lectin and placebo.

BEST MODE FOR CARRYING OUT THE INVENTION

The Inventive Compositions

The invention is directed to pharmaceutical compositions wherein theactive ingredient is a soluble, 14 kD lectin or fragment thereof. Thepreferred lectin of the invention is isolatable from HL-60 cells as wellas from human placental tissue. The lectins of this invention aresoluble, capable of binding β-galactoside-containing moietiesindependent of the presence or absence of calcium ion, capable ofstimulating hemagglutination of trypsinized rabbit erythrocytes instandard lectin assays which can be inhibited by lactose orthiogalactoside, and at least 90% homologous to the relevant portion ofpositions 2-135 of FIG. 1. One embodiment consists essentially of theamino acids shown in FIG. 1 numbered from 2-135 which contain at leastone tripeptide sequence, Asn-X-Thr or Asn-X-Ser. Preferably, thistripeptide sequence provides a glycosylation site. In native productionby HL-60 cells and placental tissue, the N-terminal methionine of theHL-60 lectin appears to be cleaved, and the remaining protein can beacetylated. Various forms of post-translational processing can beexpected depending on the cell producing the protein. The processedproteins resulting therefrom are, of course, included within the scopeof the invention.

In particular, it should be noted that unlike the prior art mammalianlectins whose protein sequences are known, a preferred embodiment ofthis invention provides a lectin with at least one glycosylation site,exemplified by Asn-Leu-Thr. Most preferably, the glycosylated tripeptidestarts at position 96 of the illustrated HL-60 lectin. The nativematerial, at least in part, apparently is not glycosylated at this site;however, when the lectin is appropriately produced in certainrecombinant hosts, the site can be glycosylated. Accordingly, theinvention provides for glycosylated forms, so long as glycosylation doesnot destroy activity.

The preferred lectins include the peptide comprising positions 2-135 ofSEQ ID NO:2 in FIG. 1 or the naturally occurring mutants or allelicvariations thereof. It is well understood that proteins produced byorganisms do not necessarily remain stable in the form studied, but thatthe genes encoding them are subject to occasional natural mutations andvariations that change one or a few amino acids; therefore, the proteinsresulting from natural mutation are included in the invention, so longas the mutation does not destroy activity. An allelic mutation changingas many as 15 amino acids is not expected to destroy the activity of thelectin. Preferably, the allelic mutation would change no more than about10 amino acids. Most preferably, only about five amino acids would bechanged by an allelic mutation.

Also included within the invention are fragments of the lectins typifiedby that shown in FIG. 1. The fragments, in order to be included withinthe scope of the invention, must exhibit the specific propertiescharacterizing the class, i.e., they must be soluble, capable of bindingβ-galactoside-containing moieties independent of the presence or absenceof calcium ion, capable of stimulating hemagglutination of trypsinizedrabbit erythrocytes in standard lectin assays which can be inhibited bylactose or thiogalactoside, and at least 90% homologous to the relevantportion of positions 2-135 of FIG. 1. While a specific minimum fragmentlength cannot now be identified, it is within ordinary routineexperimentation to assay candidate fragments for these activities. It iswell understood that only portions or fragments of a particular proteincan have the activity of the complete natively produced protein.Deletion of one or several amino acids from either the N- or C-terminusgenerally does not destroy activity; in addition, internal regions notrequired to maintain conformation or associated with the active sitescan be deleted. Whether or not a protein fragment falls within the scopeof the invention is readily determined by means of the assay systemsdescribed below for determination of ability to effect hemagglutinationboth in the presence and absence of lactose or thiodigalactoside.

HL-60 cells and placenta tissue produce the preferred. 14 kD β-galbinding lectin of the invention; this lectin is representative of theinventive class of 14-β-gal soluble lectins, one embodiment of which hasat least one glycosylation site. As used herein, the phrase “14-β-galmammalian lectin containing at least one glycosylation site” refers to aclass of peptides having the characteristics of the group of lectinsexemplified by the HL-60 lectin of FIG. 1 which contain at least onetripeptide sequence, Asn-X-Thr or Asn-X-Ser, which provides aglycosylation site.

To be included in the inventive class of 14-β-gal lectins or fragmentscontaining at least one glycosylation site, a peptide must exhibit thefollowing biological properties: For the forms which are not fragments,a molecular weight of the nonglycosylated protein is approximately 14kD. As a practical matter, the molecular weight ranges from about 12-18kD when it is measured by various techniques. The lectin or fragment iscapable of binding β-D-galactoside containing moieties (e.g., lactose).Specifically, the inventive lectin causes hemagglutination oftrypsinized rabbit erythrocytes in standard lectin assays, in which thestimulation of agglutination is inhibited by moieties containing theβ-galactoside linkage, such as lactose and thiogalactoside.Hemagglutination can occur without a reducing agent, which is capable ofmaintaining thiol groups in the reduced form, but hemagglutinationoccurs without metal ions, in particular calcium ions.

The inventive lectins and fragments have at least 40% homology with theHL-60 lectin of FIG. 1, preferably at least 75% homology, morepreferably over 90% homology and most preferably over 95% homology. Thepreferred location of the glycosylation site is at residues 96-99, as isthe case for the lectin of FIG. 1. However, the glycosylation site canbe within, at most, a four-amino acid spacing upstream or three-aminoacid spacing downstream, i.e., between residues 92 and 101 inclusive.Other preferred locations include those which contain Asn, X (any aminoacid), and Ser/Thr residues in any of the animal lectins at nonconservedregions.

The most preferred embodiment of the 14-β-gal lectins containingglycosylation sites is that of the HL-60 lectin of FIG. 1, particularlythe lectin from HL-60 cell or placental tissue sources or a lectin fromthe naturally occurring mutants and allelic variants thereof.

Glycosylated forms of this unique lectin having a molecular weight inthe range of approximately 12-18 kD are also within the scope ofthisinvention. The glycosylation of these inventive lectins can bemanipulated to provide different properties for therapeutic anddiagnostic uses.

It is known, in general, that proteins exist in a variety of essentiallyequivalent forms including the acidic and basic salts thereof, formswhich are derivatized at side-chain functional groups, forms associatedwith lipids and membranes, and other modifications made throughpost-translational processing of the cell expressing the DNA encodingthe desired lectin. All of the proteins defined above are inclusive ofthese various forms.

Preparation of the Lectins and Fragments

The lectins of the invention can be isolated from native sources,synthesized, or produced by recombinant methods. The isolation of theselectins from native sources, such as HL-60 cells or placental cells, is.described in detail in European Publication No. 337,799, published Oct.18, 1989, and is incorporated herein by reference. This publication alsodescribes the retrieval of cDNA encoding HL-60 lectin. The structure ofthe full length cDNA clone is shown herein in FIG. 1.

The lectins and fragments of the invention can be prepared, if desired,by standard solid phase or other peptide synthesis methods. This mode ofpreparation is generally considered most suited for smaller peptidefragments. Although this method is clearly within the skill of the artwith respect to the full-length lectin sequences, such as that shown inFIG. 1, part or all of the molecule can more conveniently be synthesizedusing recombinant techniques.

For recombinant production, the DNA which encodes the inventive lectinor fragment is mobilized by ligating the appropriate sequence to controlsequences regulating expression, transfecting the resulting expressionsystems into appropriate hosts, and culturing the transformed ortransfected hosts under conditions favorable for the expression of theDNA. For procaryotic systems, an intronless DNA is required; however, ineucaryotes a genomic DNA can also be used. Genomic DNA encoding theHL-60 and placental lectin and its naturally occurring mutants andallelic variants can be recovered from the HL-60 or placental genomeusing the cDNA of FIG. 1 as a probe.

The lectin-encoding sequence can be ligated into the expression systempreceded by an ATG to obtain the lectin as a mature protein.Alternatively, signal sequences known to be operable in the intendedhost, such as the penicillinase or alkaline phosphatase system inbacteria, the alpha-factor system in yeast, or various hormone signalsequences in mammalian cells can be used to effect secretion byconstructing the expression system with the DNA encoding signal inreading phase with the lectin DNA. The lectin could also be produced asa fusion protein by ligating the coding sequence into reading frame withan additional coding sequence if desired.

A variety of host systems with appropriate controls are by now wellknown in the art. For example, among procaryotic hosts, E. coli arepreferred, although other bacterial strains, such as Bacillis andPseudomonas, could be used. Suitable control systems include, but arenot limited to, promoters associated with bacterial proteins such asβ-lactamase and lactose (lac) promoter systems (Chang et al., Nature(1977) 198:1056; the tryptophan (trp) promoter system (Goeddel et al.,Nucleic Acids Res. (1980) 8:4057); and the lambda-derived p^(L) promoterand N-gene ribosome binding site system (Shimatake et al., Nature (1981)292:128).

Similarly, a variety of vectors and promoters is known for yeast systemsand includes, but is not limited to, the promoter for 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. (1980) 255:2073), the enolasegene promoter (Holland, M. J., et al., J. Biol. Chem. (1981) 256:1385),and the leu2 gene obtained from YEp 13 (Broach, J., et al., Gene (1978)8:121).

For expression in cells of higher organisms, promoters operable in suchcells include, but are not limited to, viral promoters such as the SV40promoter (Fiers et al., Nature (1978) 273:113) and promoters derivedfrom adenovirus, bovine papilloma virus, Rous sarcoma virus, and soforth. Also usable are regulatable promoters such as the metallothioneinI or metallothionein II promoters. Control sequences for retroregulationare also available such as that associated with the crystal protein geneof Bacillus thuringiensis.

Currently available also are systems for production of recombinantproteins in insect cells and in plant cells, although plant cell systemsare currently less convenient. Their inconvenience, however, is a resultof the current state of the art, and not of an inherent incompatibilitybetween this host cell system and the gene encoding the proteins of theinvention.

The appropriate coding sequences are ligated to the control sequences inoperable configuration and in suitable vectors for transfection into theintended host. The vectors include, but are not limited to, plasmids,virus particles and phages depending on the intended host and the modeof transformation. “Transformationli”, as used herein, includes allforms of causing uptake of foreign DNA by a host cell including viralinfection, transduction, conjugation or, probably most common, inductionof uptake in vitro by transfection using transfecting agents such ascalcium chloride or DEAE/dextran, depending on the host.

The transformed cells are then screened for those which contain thedesired DNA and the successful transformants are cultured underconditions which affect the expression of the coding sequences. Thelectin produced is then purified from the medium (if the constructionresults in secretion) or from the lysed cells (if the constructionresultsin an intracellular protein).

Whether the lectin is isolated from natural sources, synthesized, orproduced by recombinant methods, the lectin can be purified by standardmethods, including extraction in lactose solution followed bychromatographic procedures. Convenient chromatographic proceduresincludes chromatography on lactose sepharose gels, a sephadex S-200 HRcolumn, or a lactose-HEMA column. After using any of thesechromatography procedures, the presence of the protein in the activefractions can be easily detected by the ability of the fraction, afterremoval of the lactose, to cause hemagglutination of trypsinized rabbiterythrocytes, wherein the hemagglutination is inhibited by millimolarconcentrations of lactose or thiodigalactoside.

Antibodies Reactive with the Inventive Lectins

The lectins of the invention can be used in conventional ways to raiseantisera reactive with, and specific for, these lectins. An antibody“specific for” the referenced lectin means an antibody which isimmunoreactive with this lectin or, in some cases, with other lectins ofthe invention, but not immunoreactive with non-galactose bindinglectins. Because of the extensive homology of the FIG. 1 HL-60 lectinwith other lectins of the inventive class, polyclonal antibodies raisedagainst this lectin are likely to cross-react with other inventivelectins. However, by producing monoclonal antibodies with respect tothis lectin, antibodies specific to one particular embodiment or to aselected group of inventive lectins can be generated. In addition,antibodies specific for various glycosylated forms can also be prepared.

Antibodies can be prepared using known techniques with specificities forany particular member of the inventive 14-β-gal lectin class, includingthose with at least one glycosylation site and in nonglycosylated andespecially glycosylated forms.

In short, the antibodies within the scope of the invention are thosewhich are reactive with one or more members of the lectins of theinvention, but the antibodies are not cross-reactive with the lectinspresently known in the art. Also included in the scope of the inventionare antisera raised by any of the lectins of the invention, since theseantisera are unique to these lectins even if they contain antibodieswhich are cross-reactive in some measure with known lectins.

Uses of the Inventive Lectins

The lectins and fragments of the invention and their compositions areuseful in a range of therapeutic and diagnostic applications. Ingeneral, these peptides and proteinsare particularly useful asimmunosuppressants. The inventive lectins and fragments can be used inthe treatment of autoimmune diseases such as myasthenia gravis. Otherautoimmune diseases which are subject to treatment by these lectinsinclude rheumatoid arthritis, systemic lupus erythematosus, juvenilediabetes, and multiple sclerosis. The inventive lectins can also beuseful in controlling allergic reactions.

Since these proteins are immune system regulators, they are also usefulin the prevention of graft-versus-host disease and inhibition ofrejection of transplants in general. Thus, the inventive lectins andfragments can be administered in conjunction with various surgicaltransplantations including skin allografts, bone marrow transplants, andorgan transplants such as kidney, heart, liver or lung transplants. Whenused as an immunosuppressant, either in treating autoimmune conditionsor in preventing transplant rejection, the lectins and fragments of thepresent invention can be administered along with amounts of knowngeneral immunosuppressants that enhance their effects. Such suitablegeneral immunosuppressants include, for example, cyclophosphamide,prednisone, cyclosporin, rapamycin, other macrolide derivatives such asFK506, azathioprine, mycophenolic acid, anti-Tac, lymphocyte immuneglobulin, and OKT3 antibodies. The inventive lectins and fragmentsthereof can be administered simultaneously or sequentially with generalimmunosuppressants.

The inventive lectins are also useful in drug delivery and diagnosticapplications by carrying the chemical entity to suitable targets.Suitable targets for lectins are those cells of manmmalian subjects withgalactose-terminating ligands. These lectins are coupled to a drug, forexample, cytotoxic or therapeutic agents, or to a label, by methods inthe art. Okuda et al., Infect. Immun. (1980) 27:690-92; Samoszuk et al.,Antibody Immunoconiugates Radiopharm. (1989) 2:37-46; and Knowles etal., J. Clin. Invest. (1973) 52: 1443-52. In addition, antibodiesspecific for the inventive lectins are useful in targeting drugs orlabels to tumors, since the level of certain lectins increases on thecell surface in metastatic cancer. Anti-lectin antibodies are coupled tothe drug, for example, a cytotoxic or therapeutic agent, or label. Fordiagnostic purposes, the label coupled to the lectin or anti-lectin canbe administered to a living maaaalian subjects (in vivo use) or used inin vitro tests, for example, as part of a test kit.

While not wishing to be bound by any theory, the Inventors propose thatthe inventive lectins behave as immunomodulating agents and regulate theimmune system by binding activated lymphocytes to other activatedlymphocytes and to endothelial cells, for example on the inside of bloodvessels. Surface glycoproteins on resting lymphocytes contain terminalsialic acid residues, but activated lymphocyte glycoproteins aredesialylated to expose galactose. Hence the inventive lectin is specificfor activated T-cells and causes them to agglutinate or to adhere toendothelial cells. It is believed that these interactions may inhibit ormodify T-cell migration, e.g. extravasation from the circulation, duringinflammation.

The inventive lectin may affect the immune response by another route.Antibodies specific for the inventive lectin have been observed reactingwith human thymic tissue, particularly thymic cortical epithelial cellswhose interaction with immature cortical thymocytes is crucial indeleting auto-reactive T-cells. Although the thymus typically atrophies.early in life and is not known to play an active role in adultautoimmune pathology, administration of the inventive lectin maypossibly increase thymic deletion of autoreactive T-cells.

Formulation

For use in therapeutic applications, the lectins and fragments areformulated in a manner suitable for the desired mode of administrationusing formulation technology known in the art as described, for example,in Remington's Pharmaceutical Sciences, 17th edition, Mack PublishingCo., Philadelphia, Pa. Typical formulations for injection includeadmixture with physiological buffer for injection such as Hank'ssolution or Ringer's solution, encapsulation in liposomes or otheremulsifying agents suited for drug delivery, and the like.

A particularly preferred method of formulation provides for long termstorage of the soluble lectin of this invention in the lyophilized, orfreeze-dried, form. Lyophilization is preferably conducted in thepresence of a concentration of a carbohydrate which is effective tostabilize the lectin during the lyophilization process and at arelatively low pH of about 5. This pH appears to minimize oxidation.Also preferred is addition of a low ionic strength buffer. Suitableprotective carbohydrates include, but are not limited to,monosaccharides such as galactose; disaccharides such as lactose,maltose and sucrose; and oligosaccharides containing galactose moieties.The preferred carbohydrates are lactose and maltose. The most preferredprotective carbohydrate is lactose. Because the lyophilized product isused in a pharmaceutical composition, the protective carbohydrate mustbe physiologically and pharmaceutically acceptable at the concentrationsused. The effective concentration of the protective carbohydrate can be1-40% wt./volume but is, preferably around 5-15%, and even morepreferably around 10%.

The maintenance of the pH at about 4 to 8, preferably at about 4.5 to 6,and more preferably at about 5 discourages oxidation of cysteineresidues. It is further preferable to maintain this pH in a buffer ofrelatively low ionic strength, since the freezing point of the mixtureis then not lowered significantly. Preferably, the buffer isbicarbonate, gluconate, lactate, acetate or phosphate. Most preferably,the buffer is citrate. It is preferred that the buffer concentration beabout 5-20 mM, more preferably 7-12 mM, and most preferably about 10 mM.Other conditions of lyophilization can also be used; however, it hasbeen found that the presence of about 10% lactose in about 10 mM citrateand a pH of 5 are particularly favorable conditions.

Administration and Dosage

The dosage level and manner of administration of the lectins andfragments of the invention depends on the indication and the subject, aswell as the severity of the condition to be treated. When the fulllectin is administered, a higher dose (mg/day) is required than whenactive fragments of the lectin are administered. Some indications foruse (such as transplantation rejection, particularly full-blownrejections) require higher doses than to others (such as rheumatoidarthritis, particularly between flare-ups of the disease).

The subject who receives the inventive lectin can be a mammal, bird orother vertebrate, because the lectins of mammals, birds, eels, and fishhave been found to be related. For purposes of this invention, the termsubjects refers to mammalian subjects, including humans, farm animals,sport animals and pets. Farm animals include, but are not limited to,cows, hogs and sheep. Sport animals include, but are not limited to,dogs and horses. The category pets includes, but is not limited to, catsand dogs. Smaller animals generally require somewhat higher doses perkilogram.

Preferred dose levels range from about 0.004 mg/kg/day to about 2mg/kg/day. When the inventive lectins are used in conjunction withgeneral immunosuppressants, however, lower dosages are generallypreferred.

In general, the inventive lectins or fragments are administered in amanner suitable for peptides or proteins—i.e., by injection or by otherparenteral routes including transmembrane or transmucosal transitions.Formulations suitable for these modes of administration are wellunderstood in the art. Oral administration is always desirable, providedthe formulation permits the substantially intact lectin or fragmentthereof to survive the digestive tract and enter the bloodstream.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Prearation of the Pharmaceutical Composition

The European publication 337,799, published Oct. 18, 1989, cited above,describes in detail the isolation of 14 kD lectin from HL-60 cells orplacenta and the recombinant production of HL-60 lectin in mammalian andbacterial cells. In the examples described hereinbelow, the lectin wasprepared recombinantly in E. coli substantially as described in theabove-referenced application, and as further described by Couraud, P.O.et al., J. Biol. Chem. (1988) 264:1310-1316, also hereby incorporatedherein by reference.

Briefly, E. coli containing an expression plasmid in which the DNA to beexpressed is that shown in FIG. 1, were grown in Luria broth (LB) tostationary phase. In general, about 5 g of cells wet weight per liter ofbroth were typically obtained. The cells were then frozen and the lectinwas extracted by purification procedures.

The frozen cell samples were thawed in ice overnight or under coolrunning water to expedite the thawing. The cells were collected in abeaker and slowly mixed with a 15 mM β-mercaptoethanol (B-ME) buffer toform a thin paste having a density of approximately 0.1 to 0.3 g/ml. Thebuffer was a solution comprised of 0.02 M Tris, 0.15 M NaCl, 0.002 MEDTA, and 15 mM Mercaptoethanol having of pH of 7.5. The cells andbuffer were maintained in a cold environment. Preferably, the coldenvironment is a room that is maintained between 2 and 6° C. The cellsand buffer were mixed for a time period as long as two hours until ahomogeneous, lump-free, cell suspension results. Magnetic mixers,overhead mixers or other well known mixers or other mechanicaldisruption devices could be used. However, the overhead mixer has beenmost effective for mixing large amounts of cells.

The cell suspension was passed twice through a high pressureMicrofluidics homogenizer, operated at 15,000 psi. The homogenizer waspacked in ice to prevent heating of the cell suspension. Tris bufferedsaline (TBS) was used to recover the disrupted cell product obtainedfrom the homogenizer. The disrupted cells were next placed in an RC5Bcentrifuge (DuPont de Nemours, Wilmington, Del.) at the maximum speed(approximately 8000 rpm) for thirty minutes to separate cell debris. Thecentrifuged cell product was next decanted to separate the proteinsupernate from the cell pellets. The supernate contains the desiredlectin. However, the cell pellets can be quick frozen on dry ice forlater possible application.

The supernate was mixed with an aqueous solution containing 10% byvolume, pH 8.0, polyethylenimide (PEI). The supernate/PEI mixture wasstirred slowly in a cold environment for approximately one half hour. Amilky white precipitate formed which contains the desired nucleic acids.The mixture containing the precipitate was next placed in the centrifugeat the maximum speed (approximately 8000 rpm) for 20 to 30 minutes toremove the precipitated nucleic acids. A clear yellow/tan supernate wasobtained from this second centrifugation process. The yellow/tansupernate can be stored at 4° C. overnight before processing it throughchromatography columns the next day.

In an alternative method, PEI can be added before the firstcentrifugation step, instead of before the second centrifugation step,which can then be skipped. However, the addition of PEI before thesecond centrifugation step is preferred because it preserves the cellpellets without the added protein of the PEI precipitate. Furthermore,testing revealed that no loss of product resulted by adding the PEIbefore the second centrifugation step. However, since the secondcentrifugation step adds at least one additional hour to the totalprocess time, an alternative method of adding PEI before the firstcentrifugation step can be used to make this claimed invention in lesstime.

The next step was chromatography on lactose Sepharose gels. First, thelactose Sepharose column (Pharmacia, Uppsala, Sweden) was equilibratedwith TBS and MCE buffer. Next, the yellow/tan supernate was loaded ontothe column at 12 to 20 cm/hr. Then, the column was washed withequilibrium buffer at 20 to 25 cm/hr until UV absorbance returned tobaseline. Next, the column flow was reversed, and the buffer wash wascontinued for a time period equal to the time it would take to fillone-half of the column at the desired flow rate. The product was theneluted with 0.1 M lactose in gradient form. The gradient method ofelution is preferred for smaller quantities; however, the batch methodis preferred for larger quantities.

An alternative chromatographic procedure involves ion-exchangechromatography on a Sephadex S-200 column (Pharmacia). The column isdepyrogenated using sodium hydroxide washes of decreasing concentration.The S-200 column is next equilibrated with 10 mM citrate, having a pH of5.0. The yellow/tan supernate is loaded onto the column at a rate ofapproximately 15 cm/hr. The supernate can be loaded at a rate up to 6%by volume of the total solvent exchange. The product concentration atloading should be as high as possible to eliminate or minimize the needto further concentrate the eluted product. Immediately upon obtainingthe peak from the S-200 column, the eluted product is diluted in a 1:1ratio with sterile-filtered 10% Lactose in 10 mM Citrate having a pH of5.0.

A further alternative chromatographic procedure includes the use of aLactose-HEMA column.

The eluted product obtained from the above chromatographic procedure wasstored in small vials, with about 1 to 5 ml of the product in theappropriately sized vial. The vials were sterilized prior to filling byheating the vials to about 150° C. for a minimum of 4 hours. Any leaktight caps, such as grey split stoppers, can be used to contain theproduct within the vial.

The product was next lyophilized using a Virtis lyophilization chamberor unit. The vials containing the product were placed in the unit'schamber and frozen to −30° C. This temperature was maintained for atleast 3 hours. A vacuum was applied to the chamber and the pressure wasreduced to less than about 100 torr and preferably about 15 torr. TheVirtis unit was maintained at −30° C. and 15 torr for at least twohours. After the freezing stage, the unit and vials were brought to 0°C. and maintained at this temperature for at least two hours. Thetemperature was further increased to 30° C. in 5° C. increments over asix hour period. Once the 30° C. temperature was reached, the unit wasmaintained at 30° C. for at least two more hours. The vials were thensealed under vacuum with stoppers and aluminum seals, over which vialcaps were crimped.

The recoubinantly produced and purified lectin described above wastested to assure that it has the same electrophoretic andchromatographic mobility, the same N-terminal amino acid sequence, andthe same immunoreactivity on Western blots as that of the 14 kDinventive lectin derived from either human placenta or HL-60 cells.

EXAMPLE 2 Effect of the Inventive 14 kD Lectin on Lymphocyte Binding toEndothelial Cells

To analyze the mechanism of action of the inventive lectins asimmunomodulators, a test was devised to discover whether these lectinsbind lymphocytes and/or T-cells to endothelial cells. Thin sections oftissue containing inflamed rat brain vascular endothelium were prepared.Rat lymph node lymphocytes and human T-cells of the Jurkat line (10⁷/mlin Dulbecco's modified Eagle medium (MEM) with 5% fetal bovine serum(FBS)) were treated with 250 μg/ml of the lectin of Example 1, with thelectin plus 1 mM lactose, or with no additions and subsequently wereplaced on ice for 30 min. The lectin severely agglutinated the T-cellsand moderately clumped the rat lymphocytes. These effects were notobserved when lactose was added or when there were no additions.

Next the treated cells were layered onto the vascular endothelialsections. These treated sections were gyrated (60 rpm) on ice for 30min. Then the sections were fixed, washed and stained with toluidineblue for viewing. Both the rat lymphocytes and the T-cells bound to theendothelium of inflamed cerebral blood vessels. T-cells treated withlectin did not bind to the endothelium, possibly because the T-cellswere agglutinated and unavailable to react. Lectin strongly accentuatedthe binding of rat lymphocytes to the endothelium. Excess lactoseeliminated the lectin effect both on T-cells and lymphocytes, so thatbinding was the same as the no-lectin control. Table 1 summarizes theseresults:

TABLE 1 Binding to Inflamed Brain Endothelium Control Lectin Lectin +Lactose Rat lymphocytes + ++++ + Jurkat T-cells ++++ ± ++++

While not wishing to be bound by any particular theory, it appears thatlectin enhances binding of rat lymphocytes to vascular endothelium byreacting with both the endothelium and with the lymphocytes and servingas a molecular bridge. The lectin did not similarly bind T-cells toendothelium, possibly because the T-cells were so thoroughlyagglutinated that no free cells were available to bind the endothelium.

Sections of lymphoid organs (lymph nodes and Peyer's patch) were testedas above. Lectin had no noticeable effect on rat lymphocytes; however,because control level binding was very low, this result cannot be reliedon.

EXAMPLE 3 Cross-Reactivity of a 14 kD Lectin With Endogenous Lectin

In further characterizing the effects of the inventive lectin on the imeresponse, a test was devised to determine whether the inventive lectinis normally present in lymph nodes where it could affect thymicmaturation. First, anti-lectin antiserum was prepared by injecting theinventive recombinant lectin into rabbits. Polyclonal antiserum specificfor the inventive lectin was obtained from the immunized rabbits. Slideswere made of sections of human thymic cortex and of cultured thymicepithelial cells. These sections of human thymic tissue and culturedthymic epithelial cells were incubated with the rabbit anti-lectinantisera. The cross-reactivity was detected at a dilution of 1:1000 bylabeling the antibody-coated sections with goat anti-rabbit horseradishperoxidase and adding substrate. The thymic sections were examined bymicroscopy and had highly stained thymic cortical epithelial cells. Thecultured thymic epithelial cells were also highly stained. Theepithelial nature of the stained thymic cells was confirmed bycounterstaining with cytokeratin antibodies which are specific forepithelial cells. See FIG. 2 for the comparison of anti-lectin stainingand anti-cytokeratin staining. Thus, a protein which is immunologicallycross-reactive with the inventive recombinant lectin has been localizedto epithelial cells in human thymic tissue. In addition, proteinextracts of cultured thymic epithelial cells stained positively with therabbit-polyclonal antiserum on Western blots in an immunoreactive bandwhich comigrated with the inventive lectin.

The cDNA for the inventive lectin was used as a hybridization probeagainst thymic epithelial cell RNA. A discrete transcript was detectedand had the correct corresponding size, as shown in the representationof the electrophoresis gel in FIG. 3. Thus, it appears that thymiccortical epithelial cells have a protein with a molecular weight,antigen reactivity and DNA sequence similar to those properties of theinventive lectin.

Because interaction between thymic epithelial cells and immaturecortical thymocytes is crucial for proper selection of immunocompetentT-cells, the inventive lectin appears to affect thymocyte maturation. Inaddition, in comparison to mature T-cells, immature cortical thymocytesshow much higher levels of the galactose ligand which is recognized bythe inventive lectin. This further supports interaction of the inventivelectin with immature thymic cells, rather than an effect on matureT-cells.

Furthermore, galactose-terminating ligands are present on the cellsurfaces of migrating leukocytes and could be responsible for leukocyterecognition by lectin-containing homing receptors. Because the inventivelectins are specific for galactose ligands, the inventive lectins shouldaffect the extravasation caused by leukocyte-receptor interaction. Thiscan also be conveniently tested.

The effects of the lectin on the binding of cultured T-cells to thymicepithelial cells were further tested according to the protocol of Singeret al. (Proc, Natl. Acad. Sci. (1986) 83:6588-92). T-cell lines MOLT4and ARR (a T-cell line with sialic-acid-terminating glycoproteins) wereincubated with tritiated thymidine. Next, the cells were treated withneuraminidase (which cleaves terminal sialic acids from theglycoproteins on ARR cells and leaves terminal galactose),galactosidase, endolactosaminidase or no enzyme. After this step, thecells were incubated with and without lectin. All the treatment andcontrol groups of T-cells were layered over thymic epithelial cells,incubated and washed as described in Singer et al. Lastly, the amount ofbound T-cells was quantitated by decompositions per minute (DPM). Onlylectin-treated T-cells with terminal galactose residues appreciablybound to thymic epithelial cells, as shown in FIG. 4.

EXAMPLE 4 Assay for β-galactoside Binding Activity of Lectins

Biological activity of the inventive 14 kD lectins 1) isolated fromHL-60 cells, 2) isolated from placenta tissue or 3) prepared recorinantly from E. coli cells transfected with lectin cDNA operably linkedto a secretion signal was ascertained by agglutination of trypsinizedrabbit erythrocytes. As seen in FIG. 5, the top row shows a ConcanavalinA control with an agglutination end-point at 1.5 μg/ml. The lower sixrows show the three purified inventive 14 kD lectins incubated withvarying concentrations of completing sugars, β-lactose andthiodigalactoside, which are known to be potent inhibitors of the 14 kDplacental lectin. Thiodigalactoside inhibited agglutination of theerythrocytes at concentrations greater than 0.31 mM and β-lactoseinhibited agglutination at concentrations greater than 1.25 mM.

EXAMPLE 5 Effect of 14 kD β-gal Lectin in a Myasthenia Gravis Model

Experimental autoimmune myasthenia gravis (EAMG) is an antibody-mediatedautoimmune disease responding to acetylcholine receptors (AChR). Thiscondition in mice is a recognized model for studying the effects of theautoimmune response on neuromuscular transmission (Levi, G. et al., Eur.J. Immunol. (1983) 13:500-507; Lefvert, A. K. et al., Scand. J. Immunol.(1978) 8:525; Lefvert, A. K. et al., J. Neuro. Immunol. (1985) 9:41;Lefvert, A. K. et al., Eur. J. Immunol. (1982) 12:790). It appears thatmyasthenia gravis is mediated by T helper cells, which produceacetylcholine receptor antibodies. The disease is induced in mice byinjection of acetylcholine receptor. The acetylcholine receptor isobtained from the electric organ of the ray Torpedo marmorata andpurified by affinity chromatography on Naja naja siamensis neurotoxincrosslinked to Sephadex.

Young adult female BALB/c and C57B/6 mice (2-4 months of age) were usedbecause they differ in EAMG susceptibility. The mice were injectedsubcutaneously with 10 μg of purified acetylcholine receptor, inFreund's complete adjuvant without and with the 14 kD-β-gal lectin.

Blood was obtained after 3 days and after 1, 2, 3 and 4 weeks. To followthe development of the disease, the mice were observed daily for signsof neuromuscular dysfunction and after 10 days subjected to forcedexercise using repetitive grasping, swimming and inverted hang. Theseexercises were repeated after warming under a heat lamp at 35° C. for 5min. The results of these observations are presented in Table 2 and showthat in both strains of mice tested at higher lectin dose levels, therecombinant lectin markedly slowed neuromuscular deterioration.

TABLE 2 Mice Showing Clinical Signs of Neuromuscular Dysfunction (%)Mouse Strain BALB/c C57B/6 Receptor Only 25 68 Receptor + 2.5 μg 18 59Lectin Receptor + 12 μg 4 11 Lectin

After the observation period, the animals were sacrificed, skinned, andeviscerated; and a cholinergic receptor (AChR) extract was prepared asdescribed by Lefvert, A. K. et al., Scand. J. Immunol. supra fordetermination of total AChR content and Ig-complexed AChR content, asdescribed therein.

Briefly, to determine total AChR content, known portions of the AChRextracts were incubated with a ten-fold excess of¹²⁵I-alpha-bungarotoxin for 1 hour at 37° C. so as to label the receptorfor quantitation. The mixture was subjected to gel filtration onSephacryl G200 to separate free and bound toxin. To determine the amountof AChR complexed to IgG or IgM antibodies, the extract was incubatedwith a ten-fold excess of labeled bungarotoxin overnight at 4° C.;anti-mouse IgG or IgM was then added and the samples incubated overnightat 4° C., followed by separation of the precipitates, washing andcounting.

The results are shown in Table 3 together with the amount of recombinantlectin coadministered. These data show that single subcutaneous doses ofabout 7.5-12 μg recombinant lectin lowered both AChR loss and the amountof Ig-complexed AChR.

TABLE 3 Muscle Acetylcholine Receptor (AChR) Content in Mice Immunizedwith AChR With and Without Lectin (Determinations Made 10 Days AfterInjection) Carcass AChR Content % of AChR (mol × 10⁻¹¹) Complexed withIg Normal mice 3.9 ± 0.7 0 AChR + lectin (0.1 μg) 0.8 ± 0.4 48 ± 6.0AChR + lectin (1 μg) 1.4 ± 0.3 52 ± 2.0 AChR + lectin (5 μg) 0.9 ± 0.544 ± 5.0 AChR + lectin (15 μg) 3.5 ± 0.4  11 ± 10.7 AChR + lectin (25μg) 3.2 ± 0.6 11 ± 7.8 AChR only 1.5 ± 0.2 65 ± 2.6

EXAMPLE 6 B-Cell Repertoire of Mice Immunized with the AcetylcholineReceptor, Tetanus Toxoid or Pneumococcal Polysaccharide

The effect of the recombinant lectin on B-cell activation in BALB/c micewas also assayed using standard assays determining the number ofhybridomas secreting antibodies to the administered antigen.

Three BALB/c female mice (7-8 weeks old) were injected intraperitoneallywith 5 μg of AChR antibody, tetanus toxoid or pneumococcalpolysaccharide, respectively, in complete Freund's adjuvant. Two weekslater, the mice were injected intravenously with the same amount ofantigen in 0.15 M sodium phosphate buffer, pH 7.4. Tetanus toxoid wasobtained from the Swedish National Bacteriological Laboratory andconsisted of formalin-treated tetanus toxin (normally used forvaccination). A mixture of capsular polysaccharide extracts from 23different serotypes of Streptococcus pneumoniae (Pneumovax®) wasobtained from Merck Sharp & Dohme, Inc. (West Point, Pa.). Three othermice of the same strain were similarly immunized with these antigens incombination with 15 μg of the recombinant lectin; controls received onlythe antigens.

The animals were sacrificed 4 days after the booster injection andspleen cells (10⁸ cells) were fused with the nonsecreting B-lymphocytomacell line SP2-OAg14 (2×10⁷ cells). The cell mixture was distributed in6×96 Costar tray wells (2×10⁵ cells/well) with a feeder layer of mouseperitoneal macrophages (5×10⁵ cells/well). From the third day in cultureHAT (hypoxanthine-aminopterin-thymidine) medium was added. After 10-14days, supernatants were assayed for binding to the respective antigensused for immunization using an enzyme-linked immunosorbent assay(ELISA).

The results in Table 4 show that coadministration of lectin is effectivein reducing the number of primary clones producing antibodies againstAChR and tetanus toxoid. Coadministration of lectin reduced onlyslightly the number of hybridomas producing antibodies immoreactive withthe polysaccharide. It is believed that the B-cell response to the AChRand tetanus toxoid antigens is T-cell dependent, while that to thepolysaccharide is independent of T-cells.

TABLE 4 Number of Clones Producing Antibodies Against Torpedo Receptor,Tetanus Toxoid and Pneumococcal Polysaccharide (180 Primary Clones forEach Antigen) Antigen Antigen Experimental + Lectin Only % of ControlTorpedo 54 129 41 receptor Tetanus 79 161 48 toxoid Pneumococcal 111 14378 polysaccharide

The effect of coadministration of lectin on the B-cell repertoiremounted as a primary response to immunization was also tested directlyby antibody formation in the sera. Iminoassays for antibody content forthe various antigens were performed as follows:

For immunization with Torpedo acetylcholine receptor, microtiter wellswere coated overnight with 100 μl of a solution containing 5 μg/ml ofpurified receptor as prepared in Example 8, Part A, and incubated withserum (diluted {fraction (1/25)}) for 3 hours at 37° C. After washing,the plates were incubated for 3 hours at 37° C. with alkalinephosphatase-conjugated goat anti-mouse inrmmoglobulins. The plates werethen washed extensively and incubated for 1 hour at 37° C. withp-phenylphosphate ethanolamine buffer. The-reaction was stopped byaddition of 25 μl of 3 M NaOH, and the binding of the labeled antibodywas measured in an ELISA microreader at 405 nm. A normal mouse serumpool from more than 50 mice served as negative control. The cut-offlimit was the mean +4SD of cumulated values obtained with this normalserum pool. The results were compared to results obtained byradioimmmoassay and expressed in moles of toxin receptor precipitated by1 liter of serum, after subtraction of the mean ±4SD of cumulatedresults from a normal population (more than 50 mice).

For antibodies against mouse ACHR, antibodies were determined using asantigen a complex between a partially purified normal mouse skeletalmuscle receptor and ¹²⁵I-alpha-bungarotoxin. The results were expressedin moles of toxin receptor precipitated by 1 liter of serum, aftersubtraction of the mean +4SD of cumulated results from a normalpopulation (more than 50 mice).

For antibodies against tetanus toxoid and capsular poly-saccharides,microtiter wells were coated overnight with 100 μl of a solutioncontaining 5 μg/ml of the antigen and incubated with serum (diluted{fraction (1/200)}) for 3 hours at 37° C. After washing, the plates wereincubated for 3 hours at 37° C. with alkaline phosphatase-conjugatedgoat anti-mouse immunoglobulins. The plates were then washed extensivelyand incubated for 1 hour at 37° C. with p-phenylphosphate ethanolaminebuffer. The reaction was stopped by addition of 25 μl of 3 M NaOH, andthe binding of the labeled antibody was measured in an ELISA microreaderat 405 nm. A normal mouse serum pool from more than 50 mice served asnegative control. The cut-off limit was the mean +4SD of cumulatedvalues obtained with this normal serum pool. The values were expressedin milliabsorbance units after subtraction of the mean +4SD of thenormal serum pool.

Groups of BALB/c mice were immunized with tetanus toxoid, pneumococcalpolysaccharide, ray Torpedo receptor and mouse receptor without and withvarious doses of recombinant lectin. Blood was obtained after 3 days andagain after 1, 2, 3 and 4 weeks and antibody levels were determined asdescribed above.

The results are shown in FIGS. 6-9. As indicated, the primary immuneresponse to T-dependent antigens was significantly lowered when higherdoses of lectin (15 and 25 μg) were administered. No effect by any doseof the inventive lectin was observed for pneumococcal polysaccharide(FIG. 7), a T-independent antigen.

EXAMPLE 7 Effect of 14 kD β-gal Lectin on Experimental AutoimmuneEncephalomyelitis, An Animal Model for Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediateddisease considered to be a useful model for human paralytic anddemyelinating diseases such as multiple sclerosis (Vandenbark, A. etal., J. Immunol. (1985) 135:223). In the Lewis rat, paralytic signs ofEAE are induced about 14 days after injecting guinea pig myelin basicprotein (GPBP) in Complete Freund's Adjuvant (CFA). It has been shown byOffner, H. et al. (J. Inmunol. (1988) 144:3288) that T-cells specificfor GPBP in immunized rats recognize amino acid residues 72-89 of GPBPand have the ability to transfer both clinical signs of EAE anddelayed-type hypersensitivity reaction to GPBP to other animals.

In the studies reported in this example, EAE was induced by injection ofGPBP/CFA as described above.

In one protocol intravenous administration of lectin was started at day0 relative to 50 μg GPBP/CFA injection, with additional lectin treatmentat days 3 and 6. The severity of the disease was determined clinicallyand histologically:

“Clinical” ratings are as follows:

0=No signs of disease.

1=Flaccid tail.

2=Ataxia.

3=Hind quarter paralysis.

4=Quadriplegic/Moribund.

“Histologic” ratings are as follows:

Slides were examined for degree of inflammation with ratings as follows:

0=None;

0.5=a few infiltrating cells in the meninges;

1=meningeal infiltration, more organized and concentrated around bloodvessels;

2-4=increasing intensity of meningeal infiltration and perivascularcuffing in the CNS parenchyma.

As shown in Table 5, the 0, 3, 6-day protocol resulted in a slight delayin the onset of sickness; the sickness was less severe; the duration ofsickness was shorter; and weight loss was less.

TABLE 5 Treatment of EAE with Lectin Severity of Disease Day ofClinical/Histological Duration Loss of Onset Ratings (days) Weight (g)Group I 10 control 14.5 (3.2/−)  4 30 rats injected with buffer Days 0,3, 6 Group II 10 rats 17 (2.2/3.5) 3.0 20 injected with 250 μg lectinDays 0, 3, 6 (p < .01)

In a second protocol, pectin was first administered three days beforeimmunization with 50 μg GPBP/CFA and was followed by daily injectionsuntil day 7 after immunization. As shown in Table 6,with this protocol,the development of the disease was prevented completely.

TABLE 6 Treatment of EAE with Lectin Severity of Disease Loss of Day ofClinical/ Duration Weight DTH¹ Onset Histologic (days) (g) GPBP/PPDGroup I 20 control 14 3.3/3.5 4.5 40 26/18 rats injected with bufferDays −3, −2, −1, 0, 1, 2, 3, 4, 5, 6 Group II 10 rats 17 2.2²/—   3   20—/— injected with 250 μg lectin Days 0, 3, 6 Group III 10 rats NA  0³/0.50³ 0³   None³ 9³/4³ injected with 250 μg lectin Days −3, −2, −1,0, 1, 2, 3, 4, 5, 6 ¹DTH = delayed type hypersensitivity reaction, whichis reported as swelling × mm/100 (background subtracted) ²Differencefrom control has a p < 0.01 ³Difference from control has a p < 0.001

In an additional experiment, animals were given various doses of lectinon days 0-12, following the administration of 10 μg GPBP injectedintramuscularly. The results of these experiments are shown in FIGS. 10and 11.

FIG. 10 shows the severity of the.disease with lectin dosages rangingfrom 10-1500 μg intraperitoneally. FIG. 11 shows a summaryrepresentation of these results computed on day 12. As determined fromFIGS. 10 and 11, a daily dosage of 500 μg appears optimal forameliorating the symptoms of experimental autoimmune encephalitis.

EXAMPLE 8 Ability of Lectin to Prevent Primary Sensitization to GPBP andPPD Specifically

At the indicated times after injection of 50 μg GPBP/CFA, lymph nodesdraining the site of injection were collected and tested for aproliferative response to GPBP, purified protein derivative (PPD), andthe T-cell mitogen, Concanavalin A (ConA). Consistent with the DTH test,lymph node cells of the lectin-treated rats had low or absent responsesto both GPBP and PPD relative to control rats, especially on days 14 and21 during which EAE onset occurs. Responses to ConA in treated rats werenormal or augmented, however, indicating lack of globalimmmosuppression. These results are summarized in FIG. 12.

The lymph node cells were also unresponsive to the inventive lectin(data not shown), indicating that the lectin was not mitogenic. Inaddition, the sera of treated rats did not contain antibodies specificfor the lectin as measured by ELISA.

In a follow-up experiment, T-cell lines were selected from the draininglymph nodes. Although GPBP-specific T-cell lines could be raised fromthe GPBP/CFA immunized control group, no responses were observed to GPBPand no lines could be established from the lectin-treated rats. Inseparate experiments, lectin added to established T-cell lines had noinhibitory effect, however. Taken together, the data show that theinventive recombinant lectin is potent in preventing primarysensitization to both GPBP and PPD, but did not affect T-cell responsesgenerally, as shown by full T-cell responses to ConA.

EXAMPLE 9 Effect of the Inventive 14 kD Lectin on EAE Relapse

An additional murine model of EAE was used to demonstrate the effect otthe inventive lectin on this condition.

In mice, symptoms of EAE can be made to occur in cyclical fashion byboosting the animals with antigen following each cycle, and each cyclebecomes more severe until death ultimately occurs. This is a usefulmodel in which to determine therapeutic efficacy, since it closelymimics both the chronic disease relapses and the acute demyelinationassociated with multiple sclerosis. In this experiment, 20 female SJL/Jmice were immunized with lyophilized spinal cord extract dissolved inPBS plus CFA on days 0, 7 and 21. Ten mice were injected withrecombinant lectin, and ten mice were given buffer only. Thelectin-treated mice received 50 μg lectin on days 13 and 15intravenously in the tail vein, and an additional 100 μg i.v. daily fromday 18 through day 24. Mice were followed until death or sacrifice atday 42.

The results in FIG. 13 are mean clinical scores for control andlectin-treated mice. Lectin-treated mice had improved clinical scoresduring the first cycle, and improved survival and clinical scoresthrough the second cycle.

EXAMPLE 10 Effect of Lectin Treatment on Transplant Acceptance andRejection

The studies reported here relate to Brown Norway rat tissue transplantedinto Lewis rats. This model, a highly histo-incompatible donor/recipientpair, provides a very stringent test with predictive value in humantransplantation. Known general immunosuppressive agents showimmunosuppressive activity in these models. Several forms of this modelare used in the studies reported here.

A. Rat Heart Transplantation:

These studies were performed in the laboratory of Dr. Randy Morris ofStanford University. Whole rat hearts were grafted into host rats whichwere examined daily thereafter for signs of rejection. The treatmentgroups, dose and schedule of administration are summarized in Table 7.

TABLE 7 Heart Allograft Rejection No. Onset of Animals Dose ScheduleRejection (day) 2 1 mg/kg d − 3 to d + 9 9.8 ± .8 p < .001 3 2 mg/kg d −3 to d + 9 9.8 ± .8 12 Control d − 3 to d + 9 6.8 ± .6

A control group comprised twelve recipients treated daily i.v. only withbuffer control. The graft survival times were: 6d (x4), 7d (x7), and 8d,giving a mean survival of 6.82+/−0.6 d (SD). The final day of survivalis defined as the day on which the donor heart ceased to contract asassessed by lack of obvious palpitations and as confirmed by directvisualization at laparotomy.

Five recipients were treated with the recombinant lectin. All animalswere pretreated with 1 mg/kg i.v. lectin on days −3, −2 and −1. Thefirst two animals were then treated postoperatively with 1 mg/kg/d i.v.with lectin from day 1 until graft rejection. The second three animalswere treated in the postoperative period from day 1 until graftrejection with 2 mg/kg/d i.v. of lectin. Graft survivals were 9 and 10 dfor the first group of rats, and 9, 10 and 11 d for the second group. Nodramatic increase in survival was seen by doubling the dose of lectin ingroup two in the post-operative period. Therefore, graft survival timesof both groups were combined for statistical analysis. When graftsurvival was analyzed, lectin treatment produced a mean graft survivalof 9.8+/−0.84 d (SD), +/−0.38 d (SEM) with a 95% confidence limit. Usingthe one-tailed Mann-Whitney U-test to comare the differences between thegraft survival times for the saline and lectin-treated groups, a P valueof 0.00096 was obtained.

Thus, a small but very highly significant prolongation of heartallograft survival was achieved by treatment with lectin. In addition,there was no noticeable toxicity as might be manifested by weight loss.

B. Effect of General Immunosupressants in Combination with Lectin:

The heart transplant model of paragraph A was used. Five groups of 5Lewis rats received hearts from Brown Norway rats and were given thefollowing drugs by group, as shown in Table 8:

TABLE 8 Cyclosporin Lectin Survivors Group (mg/kg/day) (mg/kg/Day) onDay 16 A 0 2.5 0/5 B .75 1.0 3/5 C .75 2.5 5/6 D .75 5.0 5/5 E .75 0 2/3

Cyclosporin i.p. was started the day of the graft and continued daily.Recombinant lectin was started two days prior to transplant andcontinued daily.

The number of survivors on day 16 are shown in Table 8. The groupreceiving cyclosporin alone was unusually long lived, since animals onsuch therapy typically die within two weeks. At certain dosages ofcyclosporin and lectin (2.5 and 5.0 mg/kg/day), the combination therapyhad a higher survivor rate than cyclosporin or lectin administered aloneor the sum of the two survival rates. After day 20, the differencesbetween control and test groups decreased, which may be attributed to 1)the unusually longevity of the cyclosporin-treated rats and 2) theimmunogenicity of human lectin in rats.

C. Skin Allografts:

The work in this report was done in the laboratory of Drs. ArthurVandenbark and Halina Offner at the Oregon VA Medical Center. BrownNorway rat skin was transplanted onto the back of Lewis rats. Identicalgrafts were done on both sides of each animal's back and the results ofeach side (separated by commas) were shown for each parameter in Tables9 and 10. The days upon which each graft exhibited inflammation, signsof degeneration, and finally rejection are given along with whether ornot (+ or −) hair growth was restored in the tissue. A 250 μg/animaldose of lectin was administered i.v. daily on days 2-17. The resultsshow that the lectin treatment delayed rejection of the grafts by 7days, significantly reduced associated inflammation, and allowed thegrowth of hair in the grafted tissue.

The protocols for the studies reported in Tables 9 and 10 differed onlyin that the lectin was delivered for four additional days up to day 21(see Table 10).

TABLE 9 No. Mean 1 2 3 4 5 + SD Control Animals Inflammation  9,6 7,8  9,7 −,6 10,− (on day) Degeneration 10,6 8,10 10,7 13,8 10,8  (on day)Rejection 13,6 8,11 11,7 16,8 13,10 10 ± 3 (on day) Hair Growth −,− −,−−,− +,− −,− Histology 4+ 4+ 3+ 4+ 3+ rIML-1 treated Day 2 to Day 17:Inflammation −,− −,− −,− −,− −,− − (on day) Degeneration 15,16 11,1314,− −,− −,16 (on day) Rejection 16,17 13,15 16,16 18,18 18,18 17 ± 2(on day) Hair Growth +,+ +,+ +,+ +,+ +,+ + Histology 1+ 1+ 0 + 0

TABLE 10 Control Animal No. Mean 1 2 3 4 + SD Inflammation 6,− −,10 6,−6,7  (on day) Degeneration 6,11 6,10  6,12 7,11  8 + 2 (on day)Rejection 7,13 8,12 11,14 9,12 10 + 2 (on day) Hair Growth −,− −,− −,+−,− − rIML-1 treated Day 2 to Day 21: Mean 1 2 3 4 5 6 + SD Inflammation−,− −,+ −,− −,− −,− (on day) Degeneration 18,18 15,11 17,19 18,19 15,1915,18 17 ± 2 (on day) Rejection 21,21 17,13 18,20 19,22 18,23 19,19 19 ±2 (on day) Hair Growth +,+ −,+ +,+ +,+ +,+ +,+ +

The results of similar experiments conducted by Dr. Ann Kari-Lefvert atthe Karolinska Institute are reported in FIG. 14. In this experiment, 5mg/kg recombinant lectin was used on rats bearing single grafts and theday of graft rejection was noted. As shown in FIG. 14, treatment withthe inventive lectin considerably delayed rejection of the skinallograft.

EXAMPLE 11 In Vivo Effect of Lectin On Phenotypes of Selected RatLymphocytes

Table 11A shows a 28% increase in the number of rat lymph nodesuppressor T-cells (reactive with monoclonal antibody 0X8,Serotec-Bioproducts, Indianapolis, Ind.) from lectin-treated animals.

Table 11B shows a 32% increase in the number of spleen suppressorT-cells as well as a 19% and 24% decrease in the numbers of helperT-cells (w3/25 antibody reactive, Serotec-Bioproducts) and total T-cells(w3/25 antibody reactive, Serotec-Bioproducts), respectively.

TABLES 11A and 11B In Vivo Effect of Lectin-Treated Rats on Phenotypesof Selected Lymphocytes % CONTROL LECTIN CHANGE Anti- % % IN POSI- bodyF^(L) positive % F^(L) positive % TIVES 11A. Lymph Node Cells IgG 13115.9 — 129 16.4 — — W3/13 443 99.5 83.6 442 99.5 83.1 0 W3/25 216 78.162.2 230 76.5 60.1 −0  OX8 448 26.7 10.8 472 30.3 13.9 +28  OX6 1141 8.5 0 1208  10.0 0 0 OX12 340 9.4 0 316 9.9 0 0 11B. Spleen Cells IgG123 22.5 — 129 24.3 — — W3/13 334 98.5 76.0 311 85.9 61.7 −19  W3/25 19579.2 56.7 161 67.2 43.0 −24  OX8 311 35.4 12.9 323 41.2 17.0 +32  OX6720 14.6 0 375 15.6 0 0 OX12 262 14.9 0 363 15.4 0 0

EXAMPLE 12

Human blood cells were pretreated with the inventive HL-60 lectin andthen stained with fluoresceinated antibody having several differentspecificities. Control cells were not pretreated with lectin. Theresults in Table 12 show that recombinant human lectin specificallyreacts with human macrophages. No significant changes were observed forCD3+, CD4+, NK(L-A) or HLA-DR cell markers (Becton-Dickinson). However,the effect of lectin treatment on M1 and M3 macrophages was significant.The percent of positive cells was reduced by 33% and 26%, respectively,suggesting an interaction between lectin and epitopes recognized by M1and M3 antibodies.

TABLE 12 Histogram Analyses Control Lectin +FL1 +mode percent +FL1 +modepercent CD3+ 624 620 76.4 458 416 77.8 CD4+ 257 234 40 181.5 151 46.4NK(L-A) 169 157 10.8 106 94.7 10.8 HLA-DR 673 417 16.3 614 432 13.3 M1345 323 79.5 403 387 46.1 M3 575 556 70.7 549 517 45.1

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO: 1 <211>LENGTH: 507 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (50)...(457) <400> SEQUENCE: 1cttctgacag ctggtgcgcc tgcccgggaa catcctcctg gactcaatc atg gct tgt 58 ggtctg gtc gcc agc aac ctg aat ctc aaa cct gga gag tgc ctt cga 106 gtg cgaggc gag gtg gct cct gac gct aag agc ttc gtg ctg aac ctg 154 ggc aaa gacagc aac aac ctg tgc ctg cac ttc aac cct cgc ttc aac 202 gcc cac ggc gacgcc aac acc atc gtg tgc aac agc aag gac ggc ggg 250 gcc tgg ggg acc gagcag cgg gag gct gtc ttt ccc ttc cag cct gga 298 agt gtt gca gag gtg tgcatc acc ttc gac cag gcc aac ctg acc gtc 346 aag ctg cca gat gga tac gaattc aag ttc ccc aac cgc ctc aac ctg 394 gag gcc atc aac tac atg gca gctgac ggt gac ttc aag atc aaa tgt 442 gtg gcc ttt gac tga aatcagccagcccatggccc ccaataaagg cagctgcctc 497 tgctcccctg 507 <210> SEQ ID NO: 2<211> LENGTH: 135 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 2 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro GlyGlu 1 5 10 15 Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys SerPhe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His PheAsn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys AsnSer Lys 50 55 60 Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val PhePro Phe 65 70 75 80 Gln Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe AspGln Ala Asn 85 90 95 Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys PhePro Asn Arg 100 105 110 Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala AspGly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Asp 130 135

What is claimed is:
 1. A method of suppressing transplant rejection in amammalian subject comprising: administering a composition comprising anHL-60 lectin consisting of an amino acid sequence of positions 2-135 ofSEQ ID NO:2; wherein the composition is administered in an amountsufficient to suppress transplant rejection relative to transplantrejection in an untreated subject.
 2. The method of claim 1, wherein thetransplant is a skin transplant.
 3. The method of claim 1, wherein thethe composition additionally comprises a general immunosuppressant. 4.The method of claim 1, wherein the mammalian subject is a human.
 5. Themethod of claim 3, wherein the general immunosuppressant is selectedfrom the group consisting of: cyclosporin, rapamycin, other macrolidederivatives, azathioprine, prednisone, methylprednisone, CD4 antibodyand cyclophosphamide.
 6. The method of claim 1, wherein the transplantis a heart transplant.
 7. The method of claim 1, wherein the subjectreceiving the composition displays a decrease in transplant rejection ofabout twice that of an untreated subject at three weekspost-transplantation.
 8. The method of claim 1, wherein the subjectreceiving the composition displays a decrease in onset of transplantrejection of about 30% over a ten day period relative to the untreatedsubject.